Isolation and characterization of internalized glioma cell membranes.

A method for isolating plasma membranes based on the ability of cultured C6-glioma cells to phagocytize inert material such as polystyrene (latex) beads is described. The beads (phi 1.1 micron) were incubated for 16 h or 5 h. After several washings and homogenization of the cells, the beads with the surrounding membranes were isolated by use of a sucrose density gradient. The membranes were analyzed morphologically and biochemically. Morphological studies by means of light- and electron microscopy confirmed the intracellular localization of the beads. Enzymatic studies revealed that the specific activity of acid phosphatase decreased with shorter incubation periods (from 268.00 +/- 38.56 U X mg protein-1 X min-1 after 16 h to 125.12 +/- 9.10 after 5 h), whereas that of Na, K-ATPase showed the opposite trend (3.63 +/- 0.41 and 4.73 +/- 0.78 mumoles phosphate X mg protein-1 X h-1, respectively), indicating a lesser contamination with lysosomes. The main advantages of this procedure for membrane studies lie in purity and definite orientation ("inside-out") of the membranes.